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Reversible and irreversible differentiation of cardiac fibroblasts.

Driesen RB, Nagaraju CK, Abi-Char J, Coenen T, Lijnen PJ, Fagard RH, Sipido KR, Petrov VV - Cardiovasc. Res. (2013)

Bottom Line: Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation).Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile.Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Division of Experimental Cardiology, University of Leuven, KU Leuven, Campus Gasthuisberg O/N1 Box 704, Herestraat 49, Leuven B-3000, Belgium.

ABSTRACT

Aims: Differentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility.

Methods and results: Adult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures.

Conclusions: Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

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Structural adaptations of Fb cells in unrestrained 3-DCMs. (A) Fb phenotypes; Rhodamin-Phalloidin (red) marks stress fibres; nuclei stained with DAPI (blue); scale bars represent 10 µm. (a) Round phenotype, (b) dendritic phenotype without stress fibres, (c) dendritic phenotype with stress fibres, and (d)elongated phenotype with stress fibres. (B) Quantification of the cell fractions with specific phenotypes in the absence of serum. Fbs in 3-D cultures without mechanical strain and serum acquire a dendritic phenotype without stress fibres. p-MyoFb and non-p-MyoFb acquire an elongated phenotype with stress fibres. (C) Contraction of unrestrained 3-DCM by Fb cells. Volume of unrestrained 3-DCM after 3-day cultures. 3-DCMs were populated with: p-MyoFb; Fb pre-treated in 2-D cultures with SD-208 (3 µmol/L); non-p-MyoFb pre-treated with TGF-β1 (400 pmol/L); Fb pre-treated with SD-208 (3 µmol) and TGF-β1(400 pmol/L); Fb pre-treated with Y-27632 (10 µmol/L). No cells represent control 3-DCM without cells. *P < 0.05 vs. 3-DCM without cells (N = 3). Scale bars represent 5 (Aa) and 20 (Ab, c, and d) µm.
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CVT338F2: Structural adaptations of Fb cells in unrestrained 3-DCMs. (A) Fb phenotypes; Rhodamin-Phalloidin (red) marks stress fibres; nuclei stained with DAPI (blue); scale bars represent 10 µm. (a) Round phenotype, (b) dendritic phenotype without stress fibres, (c) dendritic phenotype with stress fibres, and (d)elongated phenotype with stress fibres. (B) Quantification of the cell fractions with specific phenotypes in the absence of serum. Fbs in 3-D cultures without mechanical strain and serum acquire a dendritic phenotype without stress fibres. p-MyoFb and non-p-MyoFb acquire an elongated phenotype with stress fibres. (C) Contraction of unrestrained 3-DCM by Fb cells. Volume of unrestrained 3-DCM after 3-day cultures. 3-DCMs were populated with: p-MyoFb; Fb pre-treated in 2-D cultures with SD-208 (3 µmol/L); non-p-MyoFb pre-treated with TGF-β1 (400 pmol/L); Fb pre-treated with SD-208 (3 µmol) and TGF-β1(400 pmol/L); Fb pre-treated with Y-27632 (10 µmol/L). No cells represent control 3-DCM without cells. *P < 0.05 vs. 3-DCM without cells (N = 3). Scale bars represent 5 (Aa) and 20 (Ab, c, and d) µm.

Mentions: In unrestrained 3-DCMs, we examined how low mechanical strain and a more physiological 3-D environment affected the fibroblastic phenotypes obtained in 2-D cultures. Cultures were studied in the absence of growth factors (no serum). Starting from SD-208-treated Fb populations, all fibroblastic cells are round-shaped after embedding (Figure 2A,a). One day later, 19% change their shape into dendritic cells with multiple long and thin extensions (Figure 2A,b). 16% of the cells become elongated with stress fibres (Figure 2B). Dendritic cells are distributed in the centre of the matrix, while the elongated phenotype is more frequent at the edges of the matrix. Dendritic Fbs do not contain stress fibres and show cortical F-actin, whereas elongated cells show stress fibres (Figure 2A,d). By Day 2, dendritic cells account for 34% and elongated cells 27% of the cell population (Figure 2B).Figure 2


Reversible and irreversible differentiation of cardiac fibroblasts.

Driesen RB, Nagaraju CK, Abi-Char J, Coenen T, Lijnen PJ, Fagard RH, Sipido KR, Petrov VV - Cardiovasc. Res. (2013)

Structural adaptations of Fb cells in unrestrained 3-DCMs. (A) Fb phenotypes; Rhodamin-Phalloidin (red) marks stress fibres; nuclei stained with DAPI (blue); scale bars represent 10 µm. (a) Round phenotype, (b) dendritic phenotype without stress fibres, (c) dendritic phenotype with stress fibres, and (d)elongated phenotype with stress fibres. (B) Quantification of the cell fractions with specific phenotypes in the absence of serum. Fbs in 3-D cultures without mechanical strain and serum acquire a dendritic phenotype without stress fibres. p-MyoFb and non-p-MyoFb acquire an elongated phenotype with stress fibres. (C) Contraction of unrestrained 3-DCM by Fb cells. Volume of unrestrained 3-DCM after 3-day cultures. 3-DCMs were populated with: p-MyoFb; Fb pre-treated in 2-D cultures with SD-208 (3 µmol/L); non-p-MyoFb pre-treated with TGF-β1 (400 pmol/L); Fb pre-treated with SD-208 (3 µmol) and TGF-β1(400 pmol/L); Fb pre-treated with Y-27632 (10 µmol/L). No cells represent control 3-DCM without cells. *P < 0.05 vs. 3-DCM without cells (N = 3). Scale bars represent 5 (Aa) and 20 (Ab, c, and d) µm.
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CVT338F2: Structural adaptations of Fb cells in unrestrained 3-DCMs. (A) Fb phenotypes; Rhodamin-Phalloidin (red) marks stress fibres; nuclei stained with DAPI (blue); scale bars represent 10 µm. (a) Round phenotype, (b) dendritic phenotype without stress fibres, (c) dendritic phenotype with stress fibres, and (d)elongated phenotype with stress fibres. (B) Quantification of the cell fractions with specific phenotypes in the absence of serum. Fbs in 3-D cultures without mechanical strain and serum acquire a dendritic phenotype without stress fibres. p-MyoFb and non-p-MyoFb acquire an elongated phenotype with stress fibres. (C) Contraction of unrestrained 3-DCM by Fb cells. Volume of unrestrained 3-DCM after 3-day cultures. 3-DCMs were populated with: p-MyoFb; Fb pre-treated in 2-D cultures with SD-208 (3 µmol/L); non-p-MyoFb pre-treated with TGF-β1 (400 pmol/L); Fb pre-treated with SD-208 (3 µmol) and TGF-β1(400 pmol/L); Fb pre-treated with Y-27632 (10 µmol/L). No cells represent control 3-DCM without cells. *P < 0.05 vs. 3-DCM without cells (N = 3). Scale bars represent 5 (Aa) and 20 (Ab, c, and d) µm.
Mentions: In unrestrained 3-DCMs, we examined how low mechanical strain and a more physiological 3-D environment affected the fibroblastic phenotypes obtained in 2-D cultures. Cultures were studied in the absence of growth factors (no serum). Starting from SD-208-treated Fb populations, all fibroblastic cells are round-shaped after embedding (Figure 2A,a). One day later, 19% change their shape into dendritic cells with multiple long and thin extensions (Figure 2A,b). 16% of the cells become elongated with stress fibres (Figure 2B). Dendritic cells are distributed in the centre of the matrix, while the elongated phenotype is more frequent at the edges of the matrix. Dendritic Fbs do not contain stress fibres and show cortical F-actin, whereas elongated cells show stress fibres (Figure 2A,d). By Day 2, dendritic cells account for 34% and elongated cells 27% of the cell population (Figure 2B).Figure 2

Bottom Line: Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation).Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile.Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Division of Experimental Cardiology, University of Leuven, KU Leuven, Campus Gasthuisberg O/N1 Box 704, Herestraat 49, Leuven B-3000, Belgium.

ABSTRACT

Aims: Differentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility.

Methods and results: Adult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures.

Conclusions: Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

Show MeSH
Related in: MedlinePlus